Previous results from this laboratory have indicated that the B-cell is the primary cell target responsible for the suppression of humoral immunity by TCDD. While other laboratories have confirmed that the B- cell is affected, there is not a clear understanding of TCDD's relative effects on B-cell proliferation and differentiation. This may be due to differences in both the B-cell populations studied, and the nature of the stimuli used to activate these cells and drive them through their proliferative and differentiative cycle. The proposed studies are based on three principle observations generated by our laboratory. First, the suppression by TCDD is critically dependent on the time of addition. We have determined that the suppression of the 5-day antibody response to sRBC, and of the 3-day antibody response to LPS, was maximal when TCDD was added during the first 24 hours and 3 hours of culture, respectively. Second, the suppression of the antibody response by direct exposure to TCDD is serum dependent. Most notably, we have determined that in the presence of normal mouse serum (NMS), the suppression of the antibody response to either sRBC or LPS by TCDD is Ah-dependent. Third, the suppression by TCDD of proliferation to either LPS or anti-Ig is manifested in low density B-cells; but not high density B-cells, when separated on a percoll density step gradient. We have also determine that TCDD suppressed LPS-stimulate immunoglobulin production (IgM ELISA) in low density B-cells only. Although the relative position of the high and low density B-cells was confirmed by cell cycle analysis and staining with acridine orange to be G0 and early G1, respectively, there is a paradox associated with simply concluding that the suppression by TCDD is manifested sometimes in G1. In the present investigation, we will focus exclusively on B-cells from B6C3F1 mice cultured in NMS. In SA#1, we will determine if movement into the G1 stage of the cycle is enough to confer susceptibility to suppression by TCDD, using anti-Ig-Induced proliferation as the endpoint. In SA#2, we will further characterize the ability of TCDD to "active" high density B-cells in the absence of any other stimuli. In SA#3, we will use an experimental model based on the ordered sequence of addition of B-cell growth and differentiation factors (i.e., including anti-Ig and T-cell derived factors; and either anti-Ia, as a surrogate for cognate interaction, or membranes from activated T- cells, as a more complete model of cognate interaction) to further characterize the suppression by TCDD. In SA#4, we will better define the specific temporal event targeted by TCDD - for SA#3 & 4, we will measure both proliferation and immunoglobulin production as endpoints. In SA#5, we will correlate changes in the functional endpoints with changes in cell cycle progression using cell cycle analysis by acridine orange, and changes in the expression of several select cell surface markers using immunofluorescence techniques.